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Casale, Valentina (2009) Isolamento, espansione e caratterizzazione fenotipica delle cellule staminali masenchimali da emocomponenti, sangue cordonale e sangue periferico mobilizzato. [Tesi di dottorato]

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Abstract (inglese)

In literature it has been described in the stromal component of the bone marrow the presence of a limited number of mesenchymal stem cells (MSCs) which posses multi-lineage potentiality and ability to differentiate into various tissues, such as, bone cartilage, fat, muscle, ligament, tendon and neural tissue. Plasticity of these cells could be shortly utilized in regenerative medicine for developing clinical trials in cell therapy. At the present, other human tissue are investigated as further alternative sources of MSCs.
This research project has these aims: development and optimization of isolation protocols specific for the different MSCs procurement sources, as well as cord blood (CB), cord tissue, bone marrow (BM) of patient affect by malignant empathy, mobilized peripheral blood (PB), peripheral blood from healthy donors, for example Buffy Coat (BC), or from patient affected by cutaneous sores (leuco-platelet concentrate CLP); expansion of isolated MSCs; comparison of phenotypical characterization between isolated MSCs and five different stromal cell lines HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40.
Isolation of mononuclear cells by CB, BM, mobilized PB, BC and CLP samples was carried out by two different methods: isolation technique after separation by 1.077 g/ml density gradient centrifugation (Ficoll-Histoprep) according to Boyum technique and depletion of red blood cells with ammonium chloride. Cord tissue, was, instead, cut in small fragments and seeded in culture medium. Different primary culture parameters were evaluated: time between withdrawal and processing sample, culture medium, cell concentration seeding and adhesive characteristics of culture supports after different coating treatments. Primary culture were then incubated at 37° C in an atmosphere of 95 % air- 5% CO2 as long as they were semi-confluent; cells were, then, detached by a PBS solution with trypsin 0.25% and EDTA 0.02% and seeded again for expansion. Furthermore, for 10 BC samples separated on Ficoll-Histoprep (5 donors with age among 18 to 36 years old and 5 donors with age among 50 to 56 years old) limit dilution were set up on 96 wells plates to verify MSCs growth. Observation of the cultures were carried out to an inverse microscope after 96 hours and after 7 and 14 days from their setting.
Finally, phenotypic characterization of isolated cells from BC seeded on 96 wells plate were carried out after 24 hours, 48 hours, 7 and 14 days culture time by means of immunocytochemistry. A specific antibody panel was utilized to recognize specific MSCs surface antigens.
Number of attached cells for CB, mobilized PB and CLP cultures was limited after 14 days culture time; cells seem polymorph, spherical and rarely fibroblast-like. On the contrary, in BC cultures, in particular conditions, cells immediately adhere to the support and they assume stretched morphology. After 7 days culture time, cells of BM cultures from patient affected by malignant hematological pathology show a spherical, polymorph and essentially fibroblast-like shape and they get confluent after 14 days. With regard to processed cord, after about one month of culture time cells flow together and they organize themselves as a fibroblast-like monostrate.
In vitro expansion of isolated cells from different samples was difficult, because they tightly adhere to the substrate and with only 2 BC samples out of 32 it was reached the 4th serial passing. Limit dilution did not point out cell growth from 96 hours to 2 weeks culture time and there are not significant differences among BC samples of two different age range. Immunocytochemical investigations carried out on primary cultures of BC pointed out the following antibody specificities; EGF, EGF-R, FGF, FGF-R, ADM, RAMP2, ET-1, CD33, CD73, CD105, KDR and collagen. MSCs, instead, do not express the following surface markers: CD14, CD15, CD34, CD42b, CD71, HLA-DR, STRO-1, actin, desmin, fibronectin and laminin.
These results are comparable to those obtained with stromal cell lines HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40 which can be considered as positive control. In particular, positivity for stromal mesenchymal markers CD73, CD105 besides KDR, demonstrate that isolated cells from BC are actually MSCs.
This preliminary experimentation shows some informations to optimize culture methods of MSCs isolated from sources alternative to bone marrow. As described in Literature, the results of the phonotypical characterization demonstrate that cell markers of cells isolated from BC are comparable to those of MSCs isolated from bone marrow. Therefore, BC could be an important source of adult stem cells immediately available for regenerative medicine, even the origin of these cells, their physiological-pathological meaning and their plastic potential are not yet cleared.

Abstract (italiano)

In Letteratura è stata descritta la presenza nella componente stromale del midollo osseo di un ristretto numero di cellule staminali mesenchimali (MSCs) con potenzialità multi-linea, differenziabili in tessuto osseo, cartilagineo, adiposo, tendineo, muscolare e nervoso. La plasticità di tali cellule potrebbe essere sfruttata a breve termine nell’ambito della medicina rigenerativa per lo sviluppo di protocolli clinici applicativi di terapia cellulare. Attualmente, altri tessuti del corpo umano sono indagati come ulteriore fonti alternative di MSCs.
Il programma di ricerca si propone i seguenti obiettivi: sviluppo ed ottimizzazione di protocolli di isolamento specifici per le differenti fonti di approvvigionamento delle MSCs, quali sangue cordonale (CB), tessuto cordonale, midollo osseo (BM) di pazienti affetti da empatie maligne, sangue periferico (PB) mobilizzato, sangue periferico da donatori sani, come i Buffy Coat (BC), o da pazienti affetti da piaghe cutanee (CLP, concentrato leuco piastrinico); tentativo di espansione delle MSCs isolate; caratterizzazione fenotipica delle MSCs isolate rispetto a quella delle 5 linee stromali HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40.
L’isolamento di cellule mononucleate da campioni di CB, BM, PB mobilizzato, BC e CLP è stato condotto mediante 2 metodiche: tecnica di separazione su gradiente Ficoll-Histoprep di densità 1.077 g/ml secondo la tecnica di Boyum e deplezione dei globuli rossi con ammonio cloruro. Il tessuto cordonale invece è stato tagliato in minuscoli frammenti e messo direttamente in coltura. Sono stati valutati vari parametri di coltura primaria, quali il tempo trascorso dalla raccolta del campione al suo processamento, il terreno di coltura, la concentrazione di semina e le caratteristiche adesive dei contenitori di coltura dopo vari trattamenti. Le colture primarie così allestite sono state incubate a 37°C e 5% di CO2 fino al raggiungimento della semi-confluenza; quindi le cellule sono state staccate del recipiente di coltura utilizzando una soluzione di Tripsina 0.25% ed EDTA 0.02% in PBS, e riseminate per l’espansione.
Inoltre su 10 campioni di BC separati su Ficoll-Histoprep (5 di donatori di età compresa tra 18 e 36 anni e 5 di età compresa tra 50 e 65 anni) sono state allestite 10 diluizioni limite su piastre da 96 pozzetti, per verificare la crescita delle MSCs. Le letture al microscopio ad inversione sono state eseguite dopo 96 ore, 7 giorni e 14 giorni dall’allestimento della coltura.
Infine è stata eseguita la caratterizzazione fenotipica delle cellule isolate da BC, seminate su piastre da 96 pozzetti, dopo 24 ore, 48 ore, 7 giorni e 14 giorni di coltura, mediante immunocitochimica, utilizzando un pannello di anticorpi per il riconoscimento di specifici antigeni di superficie per MSCs.
Nelle colture di CB, PB mobilizzato e CLP il numero di cellule adese dopo 14 giorni dalla semina è modesto; le cellule appaiono polimorfe, sferoidali e più raramente fibroblastoidi. Al contrario, nelle colture di BC, in particolari condizioni, le cellule aderiscono al recipiente quasi immediatamente ed assumono subito una morfologia fusata. Nelle colture di BM di pazienti affetti da emopatie maligne, le cellule presentano al 7° giorno di coltura forma sferica, polimorfa e prevalentemente fibroblastoide e vanno a confluenza al 14° giorno. Per quanto riguarda i cordoni processati, dopo circa un mese di coltura le cellule sono a confluenza e formano un monostrato di cellule fibroblastoidi.
L’espansione in vitro delle cellule isolate dai vari campioni è risultata difficoltosa, in quanto aderiscono fortemente al substrato e solo con 2 campioni di BC su 32 totali si è riusciti a raggiungere il 4° passaggio seriale. Le diluizioni limite non hanno evidenziato una crescita delle cellule dalle 96 ore alle 2 settimane di coltura e non si notano differenze significative tra i campioni di BC delle 2 diverse fasce di età.
Le indagini immunocitochimiche condotte sulle colture primarie di BC hanno evidenziato le seguenti specificità anticorpali: EGF, EGF-R, FGF, FGF-R, ADM, RAMP2, ET-1, CD33, CD73, CD105, KDR e collagene I. Invece le MSCs non esprimono i seguenti marcatori di superficie: CD14, CD15, CD34, CD42b, CD71, HLA-DR, STRO-1, actina, desmina, fibronectina e laminina. Tali risultati sono comparabili a quelli ottenuti con le linee stromali HM1 SV40, HM2 SV40, HCB1 SV40, BAEP2 WILD, BAEP2 SV40, che sono da considerare il controllo positivo. In particolare le positività per i marcatori stromali mesenchimali CD73, CD105, oltre che per KDR, dimostrano che le cellule isolate da BC sono effettivamente MSCs.
Questa sperimentazione preliminare fornisce alcune indicazioni per l’ottimizzazione dei metodi di coltura di MSCs isolate da fonti alternative al midollo osseo. Come descritto in Letteratura, i risultati della caratterizzazione fenotipica dimostrano che i marcatori cellulari delle cellule isolate da BC sono comparabili a quelli delle MSCs isolate da midollo osseo. Perciò i BC potrebbero essere un’importante fonte di cellule staminali adulte immediatamente sfruttabili in medicina rigenerativa, anche se sono ancora da chiarire l’origine di tali cellule, il loro significato fisio-patologico e il loro potenziale plastico.

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Tipo di EPrint:Tesi di dottorato
Relatore:Parnigotto, Pier Paolo
Dottorato (corsi e scuole):Ciclo 21 > Scuole per il 21simo ciclo > BIOLOGIA E MEDICINA DELLA RIGENERAZIONE > INGEGNERIA DEI TESSUTI E DEI TRAPIANTI
Data di deposito della tesi:28 Gennaio 2009
Anno di Pubblicazione:Gennaio 2009
Parole chiave (italiano / inglese):MSCs, sangue cordonale, sangue periferico mobilizzato, buffy coat
Settori scientifico-disciplinari MIUR:Area 05 - Scienze biologiche > BIO/16 Anatomia umana
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Farmaceutiche
Codice ID:1543
Depositato il:28 Gen 2009
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